Signaling by the secreted Wnt family of lipoproteins regulate many fundamental aspects of embryogenesis and tissue homeostasis. Deregulated Wnt signaling has been implicated in human diseases including birth defects, cancer, osteoporosis and degenerative disorders. The study of the mechanisms of Wnt signaling thus has critical importance for understanding basic biology and human health, and for potential disease treatment. In addition to Wnt proteins, distinct families of secreted and membrane-bound Wnt agonists and antagonists have been found that enhance or inhibit Wnt signaling during embryonic development and in physiological or pathological conditions. These Wnt modulators regulate Wnt signaling output in stage- and/or tissue-specific manner and often underlie diverse functions that Wnt pathways control, and they represent potential therapeutic targets for Wnt-related pathogenesis. The Xenopus embryo has been a powerful model system for the discovery and characterization of many families of Wnt modulators in vertebrate embryogenesis. Building on our previous success in this area, this renewal application will focus on the mechanisms and functions of two families of Wnt antagonists. Aim 1 is to study the Tiki protein, a novel Wnt-inactivating enzyme that we identified and is required for Xenopus anterior patterning. Tiki is the first example of Wnt signaling inhibition through enzymatic inactivation of the ligand, and represents the founding member of a new metalloprotease clan that is conserved from pathogenic bacteria to human. We will characterize Tiki enzymatic function, Wnt cleavage specificity, and its mechanism in regulating different Wnt signaling pathways. Aim 2 and Aim 3 are to study the molecular basis and biological functions of another conserved Wnt antagonist during Xenopus embryogenesis. The mechanism by which this Wnt antagonist operates is not fully understood, and will be investigated in Aim 2 using conventional and advanced molecular and biochemical techniques. Based on our findings that the Wnt antagonist is expressed maternally and during cleavage and gastrulation stages of Xenopus embryos, its potential roles in regulating Wnt-mediated dorsoventral and anteroposterior patterning in different developmental stages will be investigated in Aim 3. These molecular and embryological experiments together will likely provide novel insights into Wnt signaling regulation and vertebrate embryogenesis.